The Ran GTPase is required for many cellular functions, including nucleocytoplasmic trafficking, spindle assembly, nuclear assembly and cell cycle control. Ran?s nucleotide exchange factor is called RCC1, and it remains chromatin associated throughout the cell cycle. Ran?s GTPase activating protein is called RanGAP1. Ran-GTP nucleotide hydrolysis also requires a family of Ran-GTP binding proteins, which act as RanGAP1 accessory factors. This family includes the RanBP1 protein and the nucleoporin RanBP2. SUMO-1 conjugation of RanGAP1 promotes its association with the interphase nuclear pore complex (NPC) through binding to RanBP2. RanBP2 is found on kinetochores in mitosis, and RanGAP1 also concentrates at kinetochores in a microtubule and SUMO-1-dependent fashion. Our work suggests that Ran has two important roles at mitotic kinetochores. Namely, it is essential for regulation of the spindle assembly checkpoint and for assembly of microtubule fibers that attach kinetochores to spindle poles. [unreadable] We have shown that RanBP2 and RanGAP1 are targeted during mitosis as a single complex that is both regulated by and important for stable kinetochore-MT association in mitotic spindles. We have found that Crm1, a Ran-GTP-binding nuclear export receptor, localizes to kinetochores in mammalian cells. Inhibition of Crm1 using the drug Leptomycin B (LMB) causes release of RanGAP1/RanBP2 from kinetochores and the formation of spindles in which continuous MT bundles span the centromeres, indicating that their kinetochores do not maintain discrete end-on attachments to single kinetochore fibers. These findings demonstrate that proper localization of RanGAP1/RanBP2 is critical for definition of kinetochore fibers and for chromosome segregation at anaphase. Additional data supporting a role of the RanGAP1/RanBP2 complex at kinetochores have also been obtained by our collaborator (Roberge). In particular, they have identified a small molecular inhibitor, ent-15-oxokaurenoic acid (EKA), which binds to RanBP2. EKA blocks association of the mitotic motor protein CENP-E with kinetochores and inhibits chromosome movement, consistent with these a role of RanBP2 in these processes. Notably, the targeting of RanGAP1 to spindles is conserved between animals and plants, although the molecular mechanims that are utilized are completely distinct (Meier). We are currently attempting to identify kinetochore components required for the Crm1-mediated recruitment of the RanGAP1/RanBP2 complex, and to understand this mechanism at the molecular level.[unreadable] The spindle assembly checkpoint monitors spindle formation and prevents the onset of the metaphase-anaphase transition until chromosomes are correctly attached and aligned on the metaphase plate. In previous experiments, we documented that the spindle assembly checkpoint can be regulated through Ran-GTP in Xenopus egg extracts. In yeast and mammalian cells, the spindle assembly checkpoint proteins Mad1p and Mad2p localize to the NPCs during interphase, and we have examined the relationship of these proteins to the Ran pathway in budding yeast. We found that deletion of yeast MAD1 or MAD2 did not grossly affect steady-state nucleocytoplasmic trafficking or Ran localization. However, yeast with conditional mutations in the yeast Ran GTPase pathway that disrupt the concentration of Ran in the nucleus displaced Mad2p but not Mad1p from the NPC. The displacement of Mad2p in M-phase cells was correlated with activation of the spindle checkpoint. These observations demonstrate that Mad2p localization at NPCs is sensitive to nuclear levels of Ran and suggest that release of Mad2p from NPCs is closely linked with spindle assembly checkpoint activation in yeast. This is the first evidence indicating that Ran affects the localization of Mad2p to the NPC.[unreadable] Both the targeting of the RanGAP1/RanBP2 complex to mitotic kinetochores and the association of Mad1 and Mad2 to the interphase NPC suggests an intimate relationship between kinetochores and nuclear pores. Work of our collaborators (D. Forbes, B. Fontoura) has shown that the Nup107-160 complex, which includes nine nucleoporins, localizes at mitotic spindles and kinetochores in mammalian cells. In collaborative studies, they have further shown that the Nup107-160 complex is required for spindle assembly in Xenopus egg extracts. On the other hand, the capacity of egg extracts to polymerize microtubules in response to elevated Ran-GTP levels appears to be intact in the absence of the Nup107-160 complex. These findings do not exclude the possibility that the Nup107-160 complex may be an upstream regulator of Ran-GTP, but argue against the notion that it works as a Ran effector in spindle formation. Notably, the Nup107-160 complex is not required for activation of the spindle checkpoint in egg extracts, nor for checkpoint silencing by increased levels of Ran-GTP. We are extending these studies through the examination of the mitotic roles of other nucleoporin complexes, and investigation of their possible function as mitotic Ran effectors.